Miyuki Kobayashi

Tandem duplication of the FLT3 gene is found in acute lymphoblastic leukaemia as well as acute myeloid leukaemia but not in myelodysplastic syndrome or juvenile chronic myelogenous leukaemia in children

We examined mRNA expression and internal tandem duplication of the Fms‐like tyrosine kinase 3 (FLT3) gene in haematological malignancies by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and genomic PCR followed by sequencing. By RT‐PCR, expression of FLT3 was detected in 45/74 (61%) leukaemia cell lines and the frequency of expression of FLT3 was significantly higher in undifferentiated type (B‐precursor acute lymphoblastic leukaemia; ALL) than in differentiated type cell lines (B‐ALL) (P = 0.0076). Using the genomic PCR method, 194 fresh samples including 87 acute myeloid leukaemias, 60 ALLs, 32 myelodysplastic syndromes (MDSs) and 15 juvenile chronic myelogenous leukaemias (JCMLs) were examined. Tandem duplication was found in 12 (13.8%) AMLs and two (3.3%) ALLs. Sequence analyses of the 14 samples with the duplication revealed that eight showed a simple tandem duplication and six a tandem duplication with insertion. Most of these tandem duplications occurred within exon 11, and two duplications occurred from exon 11 to intron 11 and exon 12. No tandem duplications of FLT3 gene were detected in MDS or JCML. The frequency of tandem duplication of FLT3 gene in childhood AML was lower than that in adult AML so far reported. All of the 12 AML patients with the duplication died within 47 months after diagnosis, whereas two ALL patients with the duplication have survived 44 and 72 months, respectively. These two ALL patients expressed both lymphoid and myeloid antigens and were considered to have biphenotypic leukaemia. These results suggest that tandem duplication is involved in ALL in addition to AML, but not in childhood MDS or JCML, and that childhood AML patients with the tandem duplication have a poor prognosis.

Clinical significance of early T‐cell precursor acute lymphoblastic leukaemia: results of the Tokyo Children’s Cancer Study Group Study L99‐15

Early T‐cell precursor acute lymphoblastic leukaemia (ETP‐ALL) is a recently identified subtype of T‐ALL with distinctive gene expression and cell marker profiles, poor response to chemotherapy and a very high risk of relapse. We determined the reliability of restricted panel of cell markers to identify EPT‐ALL using a previously classified cohort. Then, we applied the cell marker profile that best discriminated ETP‐ALL to a cohort of 91 patients with T‐ALL enrolled in the Tokyo Children’s Cancer Study Group L99‐15 study, which included allogeneic stem cell transplantation (allo‐SCT) for patients with poor prednisone response. Five of the 91 patients (5·5%) met the ETP‐ALL criteria. There were no significant differences in presenting clinical features between these and the remaining 86 patients. Response to early remission induction therapy was inferior in ETP‐ALL as compared with T‐ALL. The ETP‐ALL subgroup showed a significantly poorer event‐free survival (4‐year rate; 40%) than the T‐ALL subgroup (70%, P = 0·014). Of note, three of four relapsed ETP‐ALL patients survived after allo‐SCT, indicating that allo‐SCT can be effective for this drug‐resistant subtype of T‐ALL.

Mutations of the RAS genes in childhood acute myeloid leukemia, myelodysplastic syndrome and juvenile chronic myelocytic leukemia

Using the polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, 12 acute myeloid leukemia (AML) cell lines and 108 fresh childhood myeloid tumor specimens, including 67 AML, 29 myelodysplastic syndrome (MDS), and 12 juvenile chronic myelocytic leukemia (JCML) were examined for mutation in H-, K-, and N-RAS genes. The mutation was found in eight of the 120 samples (6.7%), which consisted of four cell lines (33.3%) and four fresh myeloid tumors (3.7%). The frequency of the mutation in the cell lines was apparently higher than that in fresh myeloid tumors. K-RAS gene mutations were found in two of the 67 fresh AML specimens (3%). Interestingly, these two patients had 11q23 translocations. The N-RAS gene mutation was found in one of the 29 specimens (3.4%) of MDS and in one of the 12 specimens (8.3%) of JCML. All mutations were found in codon 12, 13 or 61 of the N-RAS and K-RAS genes. Frequency of mutation of RAS genes in fresh myeloid malignancies was very low. These findings suggest that mutation of RAS genes does not play an important role in the development of childhood myeloid malignancies.

Detection of chimeric mRNAs by reverse transcriptase‐polymerase chain reaction for diagnosis and monitoring of acute leukemias with 11q23 abnormalities

Recurrent translocations involving chromosome band 11q23 are often found in human acute leukemias. Recently, the MLL gene on 11q23 and 10 partner genes involved in these translocations have been cloned and characterized. We performed a reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the resultant der(11) chimeric mRNAs of the 3 types of 11q23 translocations including t(4;11), t(9;11), or t(11;19), in 14 leukemia patients with MLL gene rearrangements. At diagnosis or relapse, chimeric mRNA could be detected in all of the 4 patients with t(4;11), 2 of 3 with t(9;11), 2 of 3 with t(11;19), and 1 of 4 with unsuccessful karyotype. In 5 patients, we could monitor minimal residual disease (MRD) serially through the clinical course. One patient, in whom chi-meric mRNA was detected during complete remission (CR) just after the induction chemotherapy, relapsed within 2 months and died, while 2 patients in which chimeric mRNA was not detected remained in CR from 10-23 months. These findings suggest that RT-PCR is a useful approach for detecting which partner gene is involved in the translocation and monitoring MRD in patients with MLL gene rearrangement. Nonetheless, the clinical relevance of MRD evaluation by RT-PCR monitoring remains controversial. Long-term and prospective investigation of a larger series of patients is needed to confirm the clinical significance of monitoring MRD by RT-PCR method.

Significance of electron-dense deposits in the mitochondrial matrix of erythroid precursors in aplastic anaemia and myelodysplastic syndrome

Recently the number of long‐term survivors of aplastic anaemia has increased, with some of these cases evolving into myelodysplastic syndrome (MDS). Because it is difficult to discriminate between aplastic anaemia and hypoplastic MDS, it is unknown whether these patients have had MDS from the time of diagnosis of aplastic anaemia. Presence of ringed sideroblasts on an iron‐stained bone marrow smear is a characteristic of some cases of MDS. Amorphous electron‐dense deposits in the mitochondrial matrices of erythroid precursors observed with an electron microscope show ringed sideroblasts, and detection of this mitochondrial pathology is useful for confirming the presence of ringed sideroblasts because this mitochondrial pathology can be found not only in erythroblasts but also in reticulocytes, which is particularly useful in cases in which few erythroblasts are found. We found this mitochondrial pathology in two of nine children who had an initial diagnosis of aplastic anaemia and in three children with hypoplastic MDS. It is unknown at present whether the first two children had aplastic anaemia or hypoplastic MDS. Our results warrant further studies on more patients to confirm the significance of amorphous electron‐dense deposits in the mitochondrial matrices of erythroid precursors.

In vitro drug resistance to imatinib and mutation of ABL gene in childhood Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia.

Imatinib, the ABL kinase inhibitor, is used not only for Philadelphia chromosome-positive (Ph + ) chronic myelogenous leukemia, but also for Ph + acute lymphoblastic leukemia (ALL), although resistance to the drug tends to develop in an early stage of the clinical course. We describe a childhood refractory Ph + ALL patient in whom progressive resistance to imatinib was correlated with the appearance of a mutation in the BCR-ABL kinase domain and in vitro drug resistance to imatinib as determined by the methyl-thiazol-tetrazolium (MTT) assay. A missense mutation of T to C (Y253H) of the ABL gene was identified in the resistant clone, suggesting that this mutation may play an etiological role in the rapid loss of drug sensitivity.

Adult survivors of children’s cancer and their offspring

Abstract Background : Although it is anticipated in Japan that the number of long‐term survivors of children’s cancer will rapidly increase and that they will have children, reports of studies concerning the offspring of such survivors have come mainly from western countries. For this reason, it seems that the results of this study will be important.

Release of High Molecular Weight Neutrophil Chemotactic Activity from Human Cultured Basophilic Cells

We have shown that cultured basophilic cells, derived from human cord blood cells, release histamine and high molecular weight neutrophil chemotactic activity (HMW‐NCA), in a dose‐dependent manner following calcium ionophore and anti‐IgE challenge. The HMW‐NCA detected from human cultured basophilic cells had a similar molecular weight to that of human chopped lung challenged by anti‐IgE as determined by gel filtration chromatography. These results suggest that cultured basophilic cells might be employed for research into the chemical mediators involved in immediate hypersensitivity.